EP0318948B1 - Immunoconjugués clivables pour la délivrance et libération d'agents sous forme naturelle - Google Patents

Immunoconjugués clivables pour la délivrance et libération d'agents sous forme naturelle Download PDF

Info

Publication number
EP0318948B1
EP0318948B1 EP88119966A EP88119966A EP0318948B1 EP 0318948 B1 EP0318948 B1 EP 0318948B1 EP 88119966 A EP88119966 A EP 88119966A EP 88119966 A EP88119966 A EP 88119966A EP 0318948 B1 EP0318948 B1 EP 0318948B1
Authority
EP
European Patent Office
Prior art keywords
antibody
agent
fragment
immunoconjugate
group
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
EP88119966A
Other languages
German (de)
English (en)
Other versions
EP0318948A3 (en
EP0318948A2 (fr
Inventor
Mark D. Hylarides
Ananthachari Srinivasan
Jeffrey N. Fitzner
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Poniard Pharmaceuticals Inc
Original Assignee
Poniard Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Poniard Pharmaceuticals Inc filed Critical Poniard Pharmaceuticals Inc
Publication of EP0318948A2 publication Critical patent/EP0318948A2/fr
Publication of EP0318948A3 publication Critical patent/EP0318948A3/en
Application granted granted Critical
Publication of EP0318948B1 publication Critical patent/EP0318948B1/fr
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents

Definitions

  • the present invention relates generally to cleavable immunoconjugates that permit release of an agent in native form under mild conditions and to methods for making and using these conjugates.
  • a reoccurring problem in medicine is that, due to the lack of specificity of the agents used for treatment of illnesses, the patient is often the recipient of a new set of maladies from the therapy. This scenario is common especially in the treatment of the various forms of cancer.
  • An approach taken to circumvent the nonspecificity of the agents used to treat diseases is to couple an agent to a carrier that possesses some degree of specificity.
  • a number of molecules have been utilized as carriers in agent delivery systems, but with limited success.
  • Carrier molecules such as liposomes, proteins, and polyclonal antibodies have been used in conjunction with a broad spectrum of pharmaceutical or cytotoxic agents including radioactive compounds, agents which bind DNA, antimetabolites, agents which act on cell surfaces, and protein synthesis inhibitors.
  • Immunoconjugates are covalently bonded hybrid molecules composed of a recognition portion, such as an antibody molecule, an antibody fragment, or a functional equivalent thereof, and a biologically active portion, such as a toxin, toxin fragment, a drug, a biological response modifier, or a radioisotope.
  • Immunoconjugates have enormous potential as potent anti-tumor agents, due to the selectivity imparted to the hybrid molecules by the antibody portion of the immunoconjugate.
  • the extraordinar selectivity of antibodies or antibody fragments permits delivery of increased doses of cytotoxic, inhibitory or radiolabeled moieties to a defined population of cells.
  • the design of the compound used to link the agent to the MAb is important.
  • the linker needs to be cleavable in order to release the agent.
  • the agent is only active, or at least more potent, when none of the linker remains attached following the cleavage, the labile bond must be the one formed between the linker and the agent.
  • the type of labile bond used should be chosen on the basis of the location, i.e., inside or outside a cell, of the release-inducing factor.
  • cleavable linker groups A number of different cleavable linker groups have been described previously.
  • the mechanisms for release of an agent from these linker groups include cleavage by reduction of a disulfide bond, by irradiation of a photolabile bond, by hydrolysis of derivatized amino acid side chains, by serum complement-mediated hydrolysis, and acid-catalyzed hydrolysis. Some of these mechanisms are susceptible to release of the agent prior to having reached the specific cell, tissue or organ. Other of these mechanisms will provide faithful external delivery, however, they are inappropriate where the actual target site of the agent is inside a cell. Where an agent activates or inactivates a cell by binding to an intracellular component, the carrier-agent conjugate must be internalized and then the agent released.
  • a way to achieve internalization of a carrier-agent conjugate is to take advantage of a cell's receptor-mediated endocytosis pathway.
  • Antibodies are one example of a carrier that will bind to cell surface receptors and be internalized. Receptors which are internalized by receptor-mediated endocytosis pass through acidified compartments known as endosomes or receptosomes. Thus, the carrier-agent conjugate will be exposed transiently to an acidic pH.
  • the present invention in one aspect, provides for a method of producing cleavable immunoconjugates comprising the steps of reacting an agent with a compound having the formula (I): where: R is a chemically reactive moiety; W is a methylene, methylenoxy, or methylenecarbonyl group or combination thereof; n is 0 to 10; Y is an O, S, or NR', wherein R' is an alkyl group of C6 or less; n' is 1 to 2; and X is an H, alkyl group of C6 or less, or alkoxy group of C6 or less.
  • the agent adds to the carbon-carbon double bond, thereby forming a derivatized agent.
  • the derivatized agent is conjugated with an antibody or antibody fragment for delivery to a cell, thereby forming the cleavable immunoconjugate.
  • a variation on this method is to reverse the order of addition of an agent to the compound and of conjugation of an antibody.
  • the invention provides a cleavable immunoconjugate.
  • the immunoconjugate has the formula (II): where: A is an antibody including the linking function, or antibody fragment including the linking function; W is a methylene, methylenoxy, or methylenecarbonyl group or combination thereof; n is 0 to 10; Y is an O, S or NR', wherein R' is an alkyl group of C6 or less; n' is 1 to 2; X is H, alkyl group of C6 or less, or alkoxy group of C6 or less; and Z is an agent.
  • the present invention provides the use of said immunoconjugates in a method for delivering to the cytoplasm of a target cell an agent free of its antibody carrier.
  • a method comprises the step of administering to a mammal a diagnostically or therapeutically effective dose of a cleavable immunoconjugate having the formula (II): where: A is an antibody including the linking function, or an antibody fragment including the linking function; W is a methylene, methylenoxy, or methylenecarbonyl group or combination thereof; n is 0 to 10; Y is an O, S or NR', wherein R' is an alkyl group of C6 or less; n' is 1 to 2; X is H, alkyl group of C6 or less, or alkoxy group of C6 or less; and Z is an agent.
  • the bond joining the agent to the conjugate is cleaved, thereby releasing the agent.
  • the bond is cleavable under a variety of conditions, including mildly acidic conditions or divalent cations, and is accelerated by heat. Since the agent may be bonded via one of its nucleophilic groups directly to the linker, cleavage can result in release of native agent.
  • the present invention provides cleavable immunoconjugates and a method for producing the immunoconjugates.
  • Conjugates in which the carrier is an antibody, fragment thereof, or functional equivalent thereof have enormous potential as potent anti-tumor agents. This is due to the selectivity imparted to the conjugate by the antibody portion.
  • Theffy selectivity of antibodies permits delivery of increased doses of agents, such as those that are cytotoxic, inhibitory, radiolabeled, or biological response modifiers.
  • cleavage occurs under mildly acidic conditions. For example, subjecting a conjugate to pH 5 results in substantially complete cleavage.
  • the release mechanism may be described using as a model the example where as oxygen is the ring heteroatom.
  • An equivalent of acid is thought to initially protonate the ring O. This is followed by formation of a stable carbocation.
  • a second equivalent of acid results in the release of the agent and the formation of an aldehyde or ketone on the compound linking the agent to the antibody.
  • Another advantage of the immunoconjugates of the present invention is that cleavage results in the release of the agent without any of the linker remaining attached.
  • Popular cleavable linkers are those bifunctional reagents with a disulfide bond interposed between two reactive end groups. Cleavage of the disulfide bond by the addition of a reducing compound leaves one end of the bifunctional reagent still attached to the agent. Many agents, however, are inactivated by the permanent addition of a linker or fragment thereof to their structures.
  • the immunoconjugates of the present invention are cleaved at the bond formed between the agent and the compound linking the agent to the antibody. Therefore, the present invention provides a way of releasing an agent in native form at the target site.
  • Yet another advantage of the present invention is the ease of preparation of the linking compound, due in part to the commercial availability of the reagents needed.
  • the present invention also provides a method for producing a cleavable immunoconjugate comprising the steps of reacting an agent, capable of addition to a carbon-carbon double bond, with a compound having the formula (I): to form a derivatized agent and conjugating the derivatized agent with an antibody or fragment thereof to form the cleavable immunoconjugate.
  • R is a chemically reactive moiety.
  • the moiety may be strongly electrophilic or nucleophilic and thereby be available for reacting directly with an antibody or fragment thereof.
  • the moiety may be a weaker electrophile or nucleophile and therefore require activation prior to the conjugation with an antibody or fragment thereof. This alternative would be desirable where it is necessary to delay activation of R until an agent is added to the compound in order to prevent the reaction of the agent with R.
  • R is chemically reactive, the scenarios differ by whether following addition of an agent, R is reacted directly with an antibody or fragment thereof or is reacted first with one or more chemicals to render R capable of reacting with an antibody or fragment thereof.
  • a discussion of reactions illustrative of the activation of R is found below.
  • W in formula I is a group that functions as a "spacer arm” and may be useful to distance the antibody or fragment thereof from the agent.
  • Groups which may be used include methylene (-CH2-), methyleneoxy (-CH2-O-), methylenecarbonyl (-CH2-CO-), amino acids, or combinations thereof.
  • the number, n, of groups such as these would be typically 0 to 30 and preferably 0 to 10.
  • W, or R where n is 0, may be attached to one or the other of the ring positions designated as ⁇ and ⁇ . Because the number of methylene ring carbons at the ⁇ position is defined by n′ which may be greater than one, the ⁇ position includes additional points for attachment of a W or an R to the ring.
  • Y in formula I is a heteroatom.
  • Preferred heteroatoms include oxygen (O), sulfur (S), or nitrogen (N).
  • nitrogen is the heteroatom, it should be in the form of a tertiary amine, i.e., NR′, such as where R′ is an alkyl group of C6 or less.
  • a particularly preferred heteroatom is O.
  • the ring size of the compound may be increased above 5 by an increase in the number, n′, of ring methylene groups.
  • Preferred are 5-membered rings, such as a dihydrofuran derivative, and 6-membered rings.
  • X may be a hydrogen (H) or another substituent, preferably an alkyl group of C6 or less and an alkoxy group of C6 or less.
  • the step of reacting an agent and a compound of formula I results in the attachment of the agent to the compound via addition to the carbon-carbon double bond on the ring.
  • a derivatized agent is formed.
  • Any agent containing a group capable of reacting with the compound may be employed in the present invention.
  • Preferred agents include drugs, toxins, biological response modifiers, radiodiagnostic compounds and radiotherapeutic compounds.
  • An agent may be reacted in its native form or a derivative thereof.
  • An example of a derivative form is where an amino group on an agent is modified by reaction with a compound, such as iminothiolane, to introduce a sulfhydryl group on the agent.
  • Preferred toxins include ricin, abrin, diptheria toxin and Pseudomonas exotoxin A.
  • Preferred radionuclides for the radiodiagnostic and radiotherapeutic compounds include 99m Tc, 186/188 Re, and 123/131 I.
  • the step of conjugating may be performed by joining an antibody or fragment thereof to the derivatized agent by direct reaction with R.
  • a preparatory step it may be desirable to include before the step of conjugation a preparatory step.
  • an antibody or fragment thereof may be itself derivatized in preparation for direct reaction with R.
  • the derivatization of an antibody or antibody fragment includes reaction with any of the numerous bifunctional reagents reported in the literature.
  • R may be a carbonyl-containing group, such as an anhydride or an acid halide, or an alkyl group containing a good leaving group, e.g., a halide.
  • the latter class of compounds may be represented by alkyl X', where X' stands for the leaving group.
  • R may be a nucleophilic group, such as an amino or sulfhydryl group, which is capable of reacting with a derivatized antibody or fragment, e.g., containing a maleimide group.
  • R is a carboxyl group and is then activated. Activation of a carboxyl group includes formation of an "active ester," such as a succinimidyl ester.
  • active ester is known to refer to esters which are highly reactive in nucleophilic substitution reactions. In the present invention, the antibody or antibody fragment would be the nucleophile.
  • R is a succinimide derivative containing a protective group, such as phenylsulfonyl. Upon removal of the group, the succinimide is converted to a maleimide which is highly reactive in nucleophilic addition reactions.
  • R may be an amino, sulfhydryl, or hydroxyl group and the conversion comprises reaction with a bifunctional reagent. It will be evident to one skilled in the art that a variety of bifunctional reagents, both homobifunctional and heterobifunctional, may be employed within the present invention.
  • the antibody employed in the present invention may be an intact molecule, a fragment thereof, or a functional equivalent thereof.
  • antibody fragments are F(ab')2 , Fab', Fab, and Fv.
  • MAbs monoclonal antibodies
  • Particularly preferred MAbs are anti-TAC, or other interleukin 2 receptor antibodies; 9.2.27 and NR-ML-05 to human melanoma associated proteoglycan; NR-Lu-10 to 37-40 kilodalton pancarcinoma glycoprotein and OVB3 to an as yet unidentified antigen.
  • Genetically engineered antibodies or fragments thereof of these and other MAbs may be employed as well.
  • a variation on this method is to reverse the order of the addition of an agent and an antibody in the formation of an immunoconjugate. Specifically, first an antibody is conjugated to a compound, whose general structure is depicted above, via R and then an agent is reacted via addition to the double bond on the compound attached to the antibody.
  • an agent is reacted via addition to the double bond on the compound attached to the antibody.
  • the present invention provides cleavable immunoconjugates having the formula (II): where: W, n, Y, n', ⁇ , ⁇ , and X are defined as described above.
  • A is an antibody or an antibody fragment, and for either it includes any linking function used to attach the antibody or fragment to yield the immunoconjugate.
  • MAbs are described above.
  • Z is an agent. Any agent capable of being covalently attached to the cleavable immunoconjugate may be employed in the present invention. Typically, an agent will be bonded to the immunoconjugate by use of a sulfhydryl, amino, or hydroxyl group on the agent. The agent may be bonded directly to the immunoconjugate or indirectly with a linking function interposed between the agent and the ring. Preferred agents include those described above.
  • An additional aspect of the present invention is its use in a method for delivering to the cytoplasm of a target cell an agent free of its antibody carrier.
  • Such a method comprises the step of administering to a mammal a diagnostically or therapeutically effective dose of a cleavable immunoconjugate having the formula (II): where W, n, Y, n', ⁇ , ⁇ , X, A, and Z are defined above.
  • a preferred mammal is man.
  • Preferred MAbs and agents include those described above.
  • the agent may be diagnostically and/or therapeutically effective.
  • a preferred diagnostic agent is a compound containing 99m Tc.
  • a diagnostically effective dose of a cleavable immunoconjugate incorporating such an agent is generally from 10 to 30, typically from 15 to 25, and preferably from 0.666 GBq to 0.74 GBq (18 to 20 mCi) per 75 kg body weight.
  • a preferred therapeutic agent is a toxin, such as Pseudomonas exotoxin A.
  • a therapeutically effective dose of a cleavable immunoconjugate incorporating such as agent is generally from 1 to 100 and preferably from 1 to 10 ng per 75 kg body weight.
  • the precise dose for a particular cleavable immunoconjugate is dependent upon the antibody used, as antibodies vary with respect to the number of receptors and their affinity for the receptors, and the agent used, as toxins, for example, vary with respect to their potency. It will be evident to one skilled in the art how to determine the optimal effective dose for a particular cleavable immunoconjugate.
  • the step of administering to a mammal a diagnostically or therapeutically effective dose of a cleavable immunoconjugate having formula II sets in motion a sequence of events in vivo that results in the agent portion of the immunoconjugate being delivered free of the antibody portion to the cytoplasm of a target cell.
  • the antibody or antibody fragment portion of the immunoconjugate imparts the selectivity which permits delivery to and binding at the surface of a specific cell.
  • An immunoconjugate of formula II is susceptible to cleavage by pH less than or equal to 6.0 and the acid-catalyzed cleavage is accelerated by heating above room temperature, about 23°.
  • Example I provides the preparation of cleavable immunoconjugates utilizing 6-mercaptopurine and MAb 9.2.27.
  • Example II describes testing for the in vitro cytotoxicity and the biodistribution and toxicology of the cleavable immunoconjugates utilizing Pseudomonas exotoxin.
  • Example III discloses isolation of phenylmercaptoacetamide utilizing a derivatized agarose.
  • An activated ester form, 3 , of 2 is prepared according to the pathway depicted in Scheme III below. Specifically, the hemithioketal derivative of 6-mercaptopurine, 2 , (1.0 equivalent) is combined with 2,3,5,6 tetrafluorophenol (TFP) (1.1 equivalent) in anhydrous THF. After addition of crystalline dicyclohexyl carbodiimide (DCC) (1.1 equivalent), the mixture is stirred for 12 hours at 25°C. After removal of the precipitated dicyclohexyl urea by filtration and removal of the THF under reduced pressure, the product is obtained. After chromatography on silica gel, product 3 is obtained.
  • the monoclonal antibody 9.2.27 is directed to a melanoma-associated antigen and is prepared to according to Morgan et al., Hybridoma 1 :27-35, 1981.
  • a methanolic solution of 3 (25 ⁇ l total volume) is transferred to a vial containing buffered antibody solution (pH 8.5-10) of at least 1 mg antibody per 1 ml.
  • the conjugation mixture is warmed at 37°C for 20 minutes.
  • the modified antibody is purified either using gel permeation chromatography or small pore filtration (e.g., Centricon ultra centrifugation).
  • the carboxyl groups on PE Prior to reaction with compound 1 (Example I.A.2.), the carboxyl groups on PE are protected, e.g., by suspension of PE in O.1N methanolic HC1 at room temperature overnight. PE is then treated with a reducing agent to provide a free sulfydryl for reaction with compound 1 .
  • a mixture of compound 1 (1.0 equivalent) and PE (1.0 equivalent) in an aqueous solution containing 10% dioxane is treated with a catalytic amount of para -toluene sulfonic acid (0.01 equivalent). The resultant mixture is allowed to stir at room temperature overnight and the derivatized PE is isolated by gel filtration chromatography.
  • succinimidyl ester of derivatized PE is prepared using N-hydroxysuccinimide and a water soluble carbodiimide. After separation by gel filtration chromatography, the carboxyl groups on PE are deblocked, e.g., by hydroxylamine, and PE is conjugated to Mab 9.2.27 according to Example I.B.
  • ADP-ribosylation is measured in a cell-free system according to the method of B.G. Vanness et al., J.Biol. Chem. 255 :10717 (1980).
  • the ADP-ribosylation activity of the cleavable PE immunoconjugates in the presence and absence of dilute acid or divalent cations is compared to the activities under the same conditions of PE alone and of non-cleavable PE immunoconjugates.
  • In vitro cytotoxicity testing is performed according to the method of A.C. Morgan, Jr. et al., JNCI 78 :1101, 1987, using 3H-leucine incorporation to measure protein synthesis inhibition due to ADP-ribosylation activity by PE.
  • PE:9.2.27 conjugates two human melanoma cell lines are utilized as targets - A375 met mix (antigen-positive) and A375 I0 Primary (antigen-negative).
  • target cells are HUT 102 (antigen-positive) and CEM (antigen-negative) as discussed in D.J.P. Fitzgerald et al., J. Clin. Invest. 74 :966 (1984).
  • Conjugates are examined in two formats: (a) short exposure, wherein the conjugate is incubated with target cells for one hour at 37°C, the monolayer gently washed, and the cultures continued for up to 72 hours before the addition of 3H-leucine; and (b) long exposure, wherein the conjugate is added and the target cells exposed for the entire length of the culture period.
  • PE is radiolabeled with 125I-para-iodophenyl (PIP) as shown by D.S. Wilbur et al., J. Nucl. Med. 27 :959 (1986). This radiolabel is not subject to dehalogenation, and thereby can be used to more effectively follow the biodistribution of conjugates.
  • PIP 125I-para-iodophenyl
  • the radiolabeled PE is conjugated to a monoclonal antibody, such as 9.2.27 or anti-TAC, using the linkers of the present invention as described in Example I.
  • Animals are sacrificed at 20 hours post-injection, and organs are blotted, weighed and counted. A %dose per gram is calculated for each tissue.
  • serum half-life is estimated by retroorbital sampling of whole blood.
  • mice are administered different doses of PE-anti-TAC and PE:9.2.27 conjugates intraperitoneally to determine an LD100. Following administration of radiolabeled PE immunoconjugates, the resultant tumor localization and biodistribution of the conjugates are determined. Non-specific toxicity of PE:anti-TAC conjugates is also assessed in cynomolgous monkeys. Monkeys are monitored for liver enzyme levels, and are observed for other relevant symptoms, including appetite, presence/absence of nausea, and temperature.
  • Aminohexyl (AH)-Sepharose-4B is obtained from Pharmacia and swelling and washing of the gel is carried out according to the procedure in Affinity Chromatography - Principles and Methods , Pharmacia Publication, June 1979, pp. 22-23.
  • the solution is added to the gel and pH is readjusted if necessary during the period of coupling, which is done overnight.
  • the Sepharose-spacer- 4 conjugate is stored at 4°C.
  • a solution of S-acetylphenethylthioacetamide is treated with excess hydroxylamine in methanol:water (1:1) to yield a mixture of products and unreacted starting material, as depicted above.
  • the solution is passed through a column containing the derivatized Sepharose prepared in part A above.
  • the thiol in the mixture phenethylmercaptoacetamide
  • the thiol in the mixture is retained in the column by formation of a covalent adduct, while the other compounds in the mixture (excess hydroxyl amine, the disulfide and unreacted S-acetylphenethylthioacetamide) are eluted.
  • the desired thiol is freed from the column by passing a pH 4-5 buffer through the column.

Landscapes

  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicinal Preparation (AREA)
  • Furan Compounds (AREA)
  • Plural Heterocyclic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Claims (23)

  1. Procédé de production d'un immunoconjugué clivable, comprenant les étapes consistant :
       à faire réagir un agent, capable de présenter une réaction d'addition à une double liaison carbone-carbone, avec un composé répondant à la formule générale suivante :
    Figure imgb0023
    dans laquelle
       R représente un groupement chimiquement réactif ;
       W représente un groupe méthylène, méthylène-oxy ou méthylène-carbonyle ou une de leurs associations ;
       n a une valeur de 0 à 6 ;
       Y représente O, S ou un groupe NR', dans lequel R' représente un groupe alkyle ayant un nombre d'atomes de carbone égal ou inférieur à 10 ;
       n' est égal à 1 ou 2 ; et
       X représente H, un groupe alkyle ayant un nombre d'atomes de carbone égal ou inférieur à 6 ou un groupe alkoxy ayant un nombre d'atomes de carbone égal ou inférieur à 6 ; pour former un dérivé de cet agent ; et
       à conjuguer ledit dérivé de cet agent avec un anticorps ou un de ses fragments pour former ledit immunoconjugué clivable.
  2. Procédé suivant la revendication 1, dans lequel l'agent est choisi dans le groupe comprenant des médicaments, des toxines, des agents de modification de réponse biologique, des composés de radiodiagnostic, des composés radiothérapeutiques et leurs dérivés.
  3. Procédé suivant la revendication 2, dans lequel la toxine est choisie entre la ricine, l'abrine, la toxine diphtérique et l'exotoxine A de Pseudomonas.
  4. Procédé suivant la revendication 1, dans lequel R représente un groupe amino, un groupe sulfhydryle, un groupe hydroxyle, un groupe contenant la fonction carbonyle ou un groupe alkyle-X', dans lequel X' représente un groupe partant.
  5. Procédé suivant la revendication 1, consistant en outre, après l'étape de réaction, à transformer le groupe R du dérivé de l'agent en un groupe capable de réagir avec un anticorps ou un de ses fragments dans une préparation pour la conjugaison avec un anticorps ou un de ses fragments.
  6. Procédé suivant la revendication 1, consistant en outre, après l'étape de réaction, à transformer en un dérivé un anticorps ou un de ses fragments dans une préparation pour la conjugaison avec le dérivé de l'agent.
  7. Procédé suivant la revendication 1, dans lequel l'anticorps ou le fragment d'anticorps est un anticorps monoclonal ou un de ses fragments.
  8. Procédé suivant la revendication 1, dans lequel les fragments d'anticorps sont des fragments F(ab')₂, Fab', Fab ou Fv.
  9. Procédé suivant la revendication 1, dans lequel Y représente O, n' est égal à 1 et l'anticorps ou le fragment d'anticorps est un anticorps monoclonal ou un de ses fragments.
  10. Procédé suivant la revendication 1, dans lequel l'ordre d'addition d'un agent et d'un anticorps ou d'un de ses fragments est inversé, de sorte que l'anticorps ou un de ses fragments soit amené à réagir avec un composé suivant la revendication 1 par l'intermédiaire du groupe R et que l'agent soit ensuite conjugué par l'intermédiaire de la double liaison carbone-carbone du composé.
  11. Immunoconjugué clivable, comprenant un immunoconjugué répondant à la formule générale suivante :
    Figure imgb0024
    dans laquelle
       A représente un anticorps comprenant la fonction de jonction, ou bien un fragment d'anticorps comprenant la fonction de jonction ;
       W représente un groupe méthylène, méthylène-oxy ou méthylène-carbonyle ou une de leurs associations ;
       n a une valeur de 0 à 10 ;
       Y représente O, S ou un groupe NR', dans lequel R' représente un groupe alkyle ayant un nombre d'atomes de carbone égal ou inférieur à 6 ;
       n' est égal à 1 ou 2 ; et
       X représente H, un groupe alkyle ayant un nombre d'atomes de carbone égal ou inférieur à 6 ou un groupe alkoxy ayant un nombre d'atomes de carbone égal ou inférieur à 6 ; et
       Z représente un agent.
  12. Immunoconjugué clivable suivant la revendication 11, dans lequel l'anticorps ou le fragment d'anticorps est un anticorps monoclonal ou un de ses fragments.
  13. Immunoconjugué clivable suivant la revendication 11, dans lequel les fragments d'anticorps sont des fragments F(ab')₂, Fab', Fab ou Fv.
  14. Immunoconjugué clivable suivant la revendication 11, dans lequel l'agent est choisi entre des médicaments, des toxines, des agents de modification de réponse biologique, des composés de radiodiagnostic, des composés radiothérapeutiques et leurs dérivés.
  15. Immunoconjugué clivable suivant la revendication 14, dans lequel la toxine est choisie dans le groupe comprenant la ricine, l'abrine, la toxine diphtérique et l'exotoxine A de Pseudomonas.
  16. Immunoconjugué clivable suivant la revendication 11, dans lequel Y représente O, n' est égal à 1 et l'anticorps ou le fragment d'anticorps est un anticorps monoclonal ou un de ses fragments.
  17. Immunoconjugué clivable répondant à la formule générale suivante :
    Figure imgb0025
    dans laquelle
       A représente un anticorps comprenant la fonction de jonction, ou bien un fragment d'anticorps comprenant la fonction de jonction ;
       W représente un groupe méthylène, méthylène-oxy ou méthylène-carbonyle ou une de leurs associations ;
       n a une valeur de 0 à 10 ;
       Y représente O, S ou un groupe NR', dans lequel R' représente un groupe alkyle ayant un nombre d'atomes de carbone égal ou inférieur à 6 ;
       n' est égal à 1 ou 2 ; et
       X représente H, un groupe alkyle ayant un nombre d'atomes de carbone égal ou inférieur à 6 ou un groupe alkoxy ayant un nombre d'atomes de carbone égal ou inférieur à 6 ; et
       Z représente un agent ;
    destiné à être utilisé comme substance thérapeutique active.
  18. Immunoconjugué clivable répondant à la formule générale suivante :
    Figure imgb0026
    dans laquelle
       A représente un anticorps comprenant la fonction de jonction, ou bien un fragment d'anticorps comprenant la fonction de jonction ;
       W représente un groupe méthylène, méthylène-oxy ou méthylène-carbonyle ou une de leurs associations ;
       n a une valeur de 0 à 10 ;
       Y représente O, S ou un groupe NR', dans lequel R' représente un groupe alkyle ayant un nombre d'atomes de carbone égal ou inférieur à 6 ;
       n' est égal à 1 ou 2 ; et
       X représente H, un groupe alkyle ayant un nombre d'atomes de carbone égal ou inférieur à 6 ou un groupe alkoxy ayant un nombre d'atomes de carbone égal ou inférieur à 6 ; et
       Z représente un agent efficace à des fins de diagnostic ou des fins thérapeutiques ; destiné à être utilisé dans un procédé pour fournir au cytoplasme d'une cellule cible un agent dépourvu de son support d'anticorps.
  19. Immunoconjugué suivant la revendication 18, dans lequel l'anticorps ou le fragment d'anticorps est un anticorps monoclonal ou de ses fragments.
  20. Immunoconjugué suivant la revendication 18, dans lequel les fragments d'anticorps sont des fragments F(ab')₂, Fab', Fab ou Fv.
  21. Immunoconjugué suivant la revendication 18, dans lequel Y représente O, n est égal à 1 et l'anticorps ou le fragment d'anticorps est un anticorps monoclonal ou l'un de ses fragments.
  22. Immunoconjugué suivant la revendication 18, dans lequel l'agent est choisi entre des médicaments, des agents de modification de réponse biologique, des composés de radiodiagnostic, des composés radiothérapeutiques, des toxines et leurs dérivés.
  23. Immunoconjugué suivant la revendication 22, dans lequel le composé de radiodiagnostic ou le composé radiothérapeutique comprend un radionucléide 99mTc, ¹⁸⁶Re, ¹⁸⁸Re, ¹³¹I ou ¹²³I.
EP88119966A 1987-12-02 1988-11-30 Immunoconjugués clivables pour la délivrance et libération d'agents sous forme naturelle Expired - Lifetime EP0318948B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US12765687A 1987-12-02 1987-12-02
US127656 1987-12-02

Publications (3)

Publication Number Publication Date
EP0318948A2 EP0318948A2 (fr) 1989-06-07
EP0318948A3 EP0318948A3 (en) 1989-07-05
EP0318948B1 true EP0318948B1 (fr) 1992-08-19

Family

ID=22431215

Family Applications (1)

Application Number Title Priority Date Filing Date
EP88119966A Expired - Lifetime EP0318948B1 (fr) 1987-12-02 1988-11-30 Immunoconjugués clivables pour la délivrance et libération d'agents sous forme naturelle

Country Status (3)

Country Link
EP (1) EP0318948B1 (fr)
JP (1) JPH01294638A (fr)
DE (1) DE3873887T2 (fr)

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL106992A (en) * 1988-02-11 1994-06-24 Bristol Myers Squibb Co Noble hydrazonic history of anthracycline and methods for their preparation
US5541297A (en) * 1988-04-01 1996-07-30 Immunomedics, Inc. Therapeutic conjugates of toxins and drugs
WO1990014844A2 (fr) * 1989-06-06 1990-12-13 Neorx Corporation Sucres utilises en tant qu'elements de liaison clivables pour l'apport et la liberation d'agents sous forme naturelle
CA2065362A1 (fr) * 1989-08-24 1991-02-25 Fook-Thean Lee Anticorps marques radioactivement pour imagerie
AU636909B2 (en) * 1989-08-24 1993-05-13 Australian Nuclear Science & Technology Organisation Radio-labelled antibodies for imaging
US5776458A (en) * 1990-12-05 1998-07-07 Pharmacia & Upjohn S.P.A. Anthracycline-conjugates
GB9026491D0 (en) * 1990-12-05 1991-01-23 Erba Carlo Spa Anthracycline-conjugates
US5505931A (en) * 1993-03-04 1996-04-09 The Dow Chemical Company Acid cleavable compounds, their preparation and use as bifunctional acid-labile crosslinking agents
WO1997023243A1 (fr) * 1995-12-22 1997-07-03 Bristol-Myers Squibb Company Segments de liaison hydrazone ramifies
GB9724838D0 (en) * 1997-11-26 1998-01-21 Franks Christopher R Compositions
US6586207B2 (en) 2000-05-26 2003-07-01 California Institute Of Technology Overexpression of aminoacyl-tRNA synthetases for efficient production of engineered proteins containing amino acid analogues
US20050287639A1 (en) 2004-05-17 2005-12-29 California Institute Of Technology Methods of incorporating amino acid analogs into proteins
WO2014084110A1 (fr) * 2012-11-30 2014-06-05 株式会社糖鎖工学研究所 Lieur à chaîne glucidique liée, composé contenant un lieur à chaîne glucidique liée et substance physiologiquement active ou son sel, et son procédé de production

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS59116229A (ja) * 1982-12-24 1984-07-05 Teijin Ltd 細胞毒性複合体を活性成分とする癌治療用剤およびその製造法
US4542225A (en) * 1984-08-29 1985-09-17 Dana-Farber Cancer Institute, Inc. Acid-cleavable compound

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Phytochemistry 1979, vol. 18, pp. 666-667, Banthorpe et al. *

Also Published As

Publication number Publication date
DE3873887D1 (de) 1992-09-24
EP0318948A3 (en) 1989-07-05
JPH01294638A (ja) 1989-11-28
EP0318948A2 (fr) 1989-06-07
DE3873887T2 (de) 1993-02-04

Similar Documents

Publication Publication Date Title
US5563250A (en) Cleavable conjugates for the delivery and release of agents in native form
US5141648A (en) Methods for isolating compounds using cleavable linker bound matrices
US4981979A (en) Immunoconjugates joined by thioether bonds having reduced toxicity and improved selectivity
US5057301A (en) Modified cellular substrates used as linkers for increased cell retention of diagnostic and therapeutic agents
EP0495265B1 (fr) Linker molécules labiles aux acides
EP0318948B1 (fr) Immunoconjugués clivables pour la délivrance et libération d'agents sous forme naturelle
EP0550663B1 (fr) Anticorps modifies a temps de clairance regule
US9913921B2 (en) Pretargeting kit for imaging or therapy comprising a trans-cyclooctene dienophile and a diene
EP0510132B1 (fr) Porteurs polymeres servant a la liberation d'agents a liaison covalente
JPH01246295A (ja) 新規なリンカーを有するアントラサイクリン・イムノコンジュゲート及びその製造方法
JPH03503898A (ja) 薬剤輸送系に関する更なる改良
JPS6270377A (ja) ハプテンで修飾された診断薬および治療薬の抗体コンプレツクス
US5017693A (en) Methods for introducing a sulfhydryl amino or hydroxyl groups to a compound
EP0434765A1 (fr) Conjugues de substance de ciblage et d'agent diagnostique/therapeutique comportant des liaisons de base de schiff.
AU4424589A (en) Vasopermeability-enhancing conjugates
NO881077L (no) Kjemo-radio-immuno-konjugater.
US6022522A (en) Rigid bifunctional chelating agents
EP1560600B1 (fr) Procede de fonctionnalisation selective et quantitative de fragments fab d'immunoglobuline, composes conjugues obtenus au moyen de ce procede et leurs compositions
Arano et al. A novel bifunctional metabolizable linker for the conjugation of antibodies with radionuclides
US5155210A (en) Methods of conjugating actinomycin d
US6007817A (en) Vasopermeability enhancing immunoconjugates
WO1996039124A1 (fr) Systemes polymeres d'affinite utiles pour l'apport de substances cytotoxiques et d'autres composes a des foyers pathologiques
EP0476408A1 (fr) Conjugaison chimique de morpholino-anthracyclines avec des anticorps
AU730388B2 (en) Antibodies with reduced net positive charge
Hawkins et al. Delivery of radionuclides to pretargeted monoclonal antibodies using dihydrofolate reductase and methotrexate in an affinity system

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

PUAL Search report despatched

Free format text: ORIGINAL CODE: 0009013

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): DE FR GB IT SE

AK Designated contracting states

Kind code of ref document: A3

Designated state(s): DE FR GB IT SE

17P Request for examination filed

Effective date: 19891123

17Q First examination report despatched

Effective date: 19901004

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): DE FR GB IT SE

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRE;WARNING: LAPSES OF ITALIAN PATENTS WITH EFFECTIVE DATE BEFORE 2007 MAY HAVE OCCURRED AT ANY TIME BEFORE 2007. THE CORRECT EFFECTIVE DATE MAY BE DIFFERENT FROM THE ONE RECORDED.SCRIBED TIME-LIMIT

Effective date: 19920819

Ref country code: SE

Free format text: THE PATENT HAS BEEN ANNULLED BY A DECISION OF A NATIONAL AUTHORITY

Effective date: 19920819

Ref country code: FR

Effective date: 19920819

REF Corresponds to:

Ref document number: 3873887

Country of ref document: DE

Date of ref document: 19920924

EN Fr: translation not filed
PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

26N No opposition filed
PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 19941026

Year of fee payment: 7

Ref country code: GB

Payment date: 19941026

Year of fee payment: 7

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GB

Effective date: 19951130

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 19951130

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DE

Effective date: 19960801